SUMMARY Respiratory Syncytial Virus (RSV) is the single largest viral cause of pediatric bronchiolitis and pneumonia, with a high worldwide mortality, and there is no safe and effective vaccine. Ever since the encounter with vaccine- enhanced disease (VED) during a formalin-inactivated RSV vaccination trial in the 1960s, it has been enormously challenging to impart both sufficient safety and efficacy in a single vaccine. For RSV-nave children, live-attenuated vaccines are an important focus, because inactivated and subunit vaccines are poor at inducing cell-mediated immunity which contributes to VED. Moreover, live vaccines can induce broad systemic and local immunity. Thus, for RSV-nave individuals a live vaccine approach is an attractive option, provided the vaccine itself is sufficiently safe and cannot revert to a more aggressive phenotype. It has recently been recognized that the viral fusion (F) protein is unstable and readily shifts to the post-fusion conformation during purification or vaccine preparation. As a result, a large proportion of vaccine-induced antibodies (Abs) target the post-fusion form, which is functionally obsolete. To avoid induction of anti- post- fusion F Abs, McLellan et al were able to genetically stabilize the pre-fusion form (referred to as FPRE), thereby greatly increasing neutralizing capacity of anti-F Abs, when given as a protein vaccine. However, subunit vaccines are deemed unsafe for the RSV-nave target population. Thus, to protect RSV-nave children there is a need to place the advantageous pre-fusion F concept in the context of a balanced immune response that does not cause VED, which could be achieved by expressing FPRE from a live-attenuated virus. However, stabilization of FPRE renders it non-functional and a virus solely expressing FPRE is not viable. We previously reported a baculovirus GP64 based complementation system that can overcome this problem, as it uniquely allows generation of infectious F-deleted or F-compromised viruses from cDNA in GP64-expressing cells. These GP64-pseudotyped viruses could be amplified to high titer and were significantly more temperature stable than wildtype RSV. Due to replacement of functional F with trans-complemented GP64, the viruses are infectious but self-limited and cannot spread beyond initially infected cells, thus constituting an attractive live-attenuated platform. We have in vivo preliminary data using a similar single-round RSV vaccine that suggests such vaccines are efficaceous. The goal of this proposal is to exploit the GP64 system to generate a live RSV which solely expresses the pre- fusion F form. Our hypothesis is that replacing the native, functional, F gene with a gene encoding a pre-fusion stabilized F in a live virus, will drive the anti-F response toward the pre-fusion F form, and will induce a balanced response that includes cell-mediated immunity and avoids VED. We will test this hypothesis through two aims: 1) Generate and in vitro characterize a live-attenuated RSV lacking a functional F copy and expressing instead a conformationally stabilized pre-fusion F; and 2) Characterize immune properties of RSV expressing FPRE in vivo to assess safety and efficacy. If successful, the vaccine will exceed previous formulations in inducing a broadly efficaceous yet safe immune response for the RSV-nave target population.